The present invention, in some embodiments thereof, relates to therapy and diagnosis (theranostic) and, more particularly, but not exclusively, to polymeric systems in which a labeling moiety (e.g., a fluorescent or fluorogenic moiety) or a labeling moiety and a therapeutically active agents are attached to a polymeric backbone, and to uses thereof in diagnostic and theranostic applications.
In the past few years, tremendous efforts have been employed in monitoring cancer treatment, detecting response to drugs and measuring real-time accumulation of the drug within the tumor. Numerous nanocarrier systems have been developed (e.g., polymers, liposomes, micelles, dendrimers, etc.) and studied as delivery vehicles for anticancer drugs to improve the drugs' biodistribution, solubility, and half-life, and thus to exhibit enhanced efficacy and reduced toxicity. Clinically-available fluorescence-based imaging contrast agents (e.g., indocyanine green and fluorescein) hold many of the limitations attributed to chemotherapeutic agents, including low molecular weight, short half-life and poor selectivity. Consequently, monitoring slow processes, such as drug accumulation at the tumor site, is challenging.
Combining therapeutic and diagnostic modalities on the same delivery system, thereby forming a theranostic (therapy and diagnostic) nanomedicine, may overcome these limitations, while enabling simultaneous monitor and treatment of angiogenesis-dependent diseases, like cancer [Kelkar, S. S. and T. M. Reineke, Theranostics: Combining Imaging and Therapy. Bioconjug Chem, 2011. 22(10): p. 1879-1903]. Information obtained from theranostic nanomedicine is exploited for fine tuning the therapeutic dose, while monitoring the progression of the diseased tissue, treatment efficacy and delivery kinetics [Janib et al. Adv Drug Deliv Rev, 2010. 62(11): p. 1052-1063; McCarthy, J. R., The future of theranostic nanoagents. Nanomedicine, 2009. 4(7): p. 693-695]. This, from a clinical prospective, should enhance early diagnosis and treatment and may decrease drugs under- or over-dosing, resulting in a more personalized treatment.
Among different imaging modalities (e.g., radiography, magnetic resonance imaging and ultrasound), optical imaging holds several advantages. Fluorescent molecular probes are highly sensitive, possess a high spatial resolution, enable simultaneous multicolor imaging and specificity, by signal activation in the tissue of interest, they may possess high target to background ratio (TBR), and are relatively inexpensive. Furthermore, they do not hold long term health risks, like other commonly-used computed tomography (e.g., PET—positron emission tomography and SPECT-single-photon emission computed tomography), which expose the patient to ionizing radiation.
An ideal theranostic nanomedicine system should hold (i) long circulation time in the body, (ii) high specificity to the target tissue, (iii) an efficient release mechanism, (iv) an imaging probe that enables monitoring its activity, (v) deep tissue penetration, and (vi) high target-to-background (TBR) ratio. High specificity can be obtained via passive targeting, by exploiting the enhanced permeability and retention (EPR) effect or via an additional functional targeting moiety.
In contrast to thin layer imaging of cells or surfaces, the signal from fluorescent probes in vivo is impeded by the emitted fluorescence from tissues and biomolecules (e.g., water, melanin, proteins and hemoglobin), which absorb photons in the wavelengths range of 200-650 nm (i.e., low signal-to-noise ratio). In addition, tissues contribute to reflection, refraction and scattering of incident photons, thus increasing the background and blur of the obtained image. The ‘imaging wavelength window’ left for intravital imaging in order to overcome these obstacles is at the near infra-red (NIR) range (i.e., 650-1450 nm). In this range, auto-fluorescence is minimal and scattering of light is reduced, enabling deep tissue penetration and facilitating non-invasive monitoring.
One way to maximize the signal from the target and to minimize the signal from background (i.e., high TBR ratio), is the use of activatable optical probes. The fluorescent signal is silenced/“OFF” under physiological conditions, and is turned-ON at a designated site and/or under specific conditions [Lee et al., Activatable molecular probes for cancer imaging. Vol. 10. 2010. 1135-44].
Although numerous classes of Turn-ON optical probes have been described in the literature for detection of chemical and biological factors [Karton-Lifshin, N., et al., J Am Chem Soc, 2011. 133(28): p. 10960-5; Kobayashi, H., et al., Chem Rev, 2010. 110(5): p. 2620-40; Lee, S., et al., Chem Commun (Camb), 2008(36): p. 4250-60; Redy-Keisar, O., et al., Nat Protoc, 2014. 9(1): p. 27-36; Weinstain, R., et al., Chem Commun (Camb), 2010. 46(4): p. 553-5], to this point, most polymer-based theranostic nanomedicines studies utilize an ‘always ON’ theranostic systems. In these systems, a fluorescent signal is obtained from the background and desired site at once, resulting in low TBR.
Among methods used to obtain a selective Turn-ON mechanism, Förster resonance energy transfer (FRET) is the most common and efficient. Using FRET technique to monitor drug release, two types of fluorophores are incorporated into the core of drug-carrying nanoparticles and serve as energy donors and acceptors. In this process, following excitation of the donor, the acceptor will absorb the emission energy of the donor and will turn off the fluorescent signal. The donor and the acceptor are required to have overlapping emission and absorbance spectra, as well as close proximity between them. A FRET-based probe is turned-ON upon distance that results in the diffusion of the donor fluorophore away from the acceptor, and generation of a measurable fluorescent signal [Lee et al. 2010 supra; Johansson, M. K., et al., Journal of the American Chemical Society, 2002. 124(24): p. 6950-6956]. This process includes two approaches, fluorophore-fluorophore (self-quenching) and fluorophore-quencher activation. The donor is always a fluorophore, however the acceptor can be either a quencher—a dye with no native fluorescence (FRET) or a second fluorophore (self-quenching) [Redy, O., et al., Org Biomol Chem, 2012. 10(4): p. 710-5].
In the fluorophore-fluorophore (self-quenching) approach, excited fluorophores of similar type absorb the energy from each other that would otherwise have led to an emitted photon, thus reducing the fluorescence of the entire compound. This can occur when the excitation and emission peaks overlap or when the Stokes shift is small, like in the case of Cy5. Hence, the fluorophore can serve as a quencher and adsorb the excitation energy. Under these circumstances the emitted energy from one fluorophore is absorbed by another fluorophore (self-quenching) [Melancon, M. P., et al., Pharm Res, 2007. 24(6): p. 1217-24].
Self-quenching involving only fluorophores may still yield weak fluorescence even in the quenched state. A second alternative to fluorophore-fluorophore quenching, is to use a fluorophore-quencher combinations in which the quencher is non-fluorescent and plays as the acceptor, whereas the donor is a fluorophore. When a FRET fluorophore-quencher process occurs, the excited fluorophore can transfer its emission energy to the nearby quencher [Redy, O., et al., Org Biomol Chem, 2012. 10(4): p. 710-5].
Optical imaging in the near-infrared (NIR) range enables detection of molecular activity in vivo clue to high penetration of NIR photons through organic tissues and low auto-fluorescence background. Cyanine dyes are widely employed as fluorescence labels for NIR imaging, since they are compounds with large extinction coefficient and relatively high quantum yield.
In order to generate a Turn-ON system for a cyanine molecule, a FRET (fluorescence resonance energy transfer) approach is usually applied. In such approach, the cyanine dye and a quencher are attached through a specific linker to obtain a quenched fluorophore. A linker, which is cleaved by a specific enzyme, separates the fluorophore from the quencher and thus, turn-ON its fluorescence signal. Exemplary such FRET-based probes are described in Redy, O., et al., Org Biomol Chem, 2012. 10(4): p. 710-5, which is incorporated by reference as if fully set forth herein. An alternative approach, to turn OFF and ON a fluorophore, could be achieved by disrupting the pull-push conjugated π-electron system of the dye. Such a concept, referred to as internal Charge Transfer (ICT) probe, is described in WO 2012/123916, which is incorporated by reference as if fully set forth herein, and in Kisin-Finfer E., et al., 1; 24(11):2453-8; Bioorg Med Chem Lett. 2014, which is also incorporated by reference as if fully set forth herein.
Additional background art includes Jones et al. Langmuir, 2001, 17 (9), pp 2568-2571; U.S. Patent Application Publication No. 20120122734; Theodora Krasia-Christoforou and Theoni K. Georgiou, J. Mater. Chem. B, 2013, 1, 3002-3025; Morton et al., Biomaterials. 2014 April; 35(11): 3489-3496; and Luk and Zhang, Appl. Mater. Interfaces 2014, 6, 21859-21873.